Metabolic dysfunction in obesity and type 2 diabetes (T2D) involves insulin resistance in multiple tissues. Recently key roles in metabolic regulation have been identified for factors produced by and secreted from adipose tissue (termed adipokines) that can either induce insulin resistance or improve insulin sensitivity. Less attention has been paid to factors produced by skeletal muscle (myokines) that could also influence insulin sensitivity, as well as impact adipose tissue (AT) accumulation and function. The hypothesis to be tested is: Skeletal muscle produces and releases factors (myokines) that can act in an autocine manner on muscle to augment or impede metabolism and insulin action as well as act in an endocrine manner to influence AT metabolism and accumulation. Muscle from obese type 2 diabetic individuals produces more of the myokines that induce insulin resistance and stimulate fat accumulation. There are 3 Specific Aims.1) Determine the ability of human skeletal muscle to produce and secrete GRO, IL-8, and IL-15 and regulation of this production by inflammatory and anti- inflammatory signals. 2) Establish the autocrine effects of these specific myokines on glucose metabolism and insulin sensitivity in skeletal muscle, and the signaling pathways involved. Are diabetic muscle cells more or less sensitive to the actions of these myokines? 3) Evaluate the effects of specific myokines on the differentiation and apoptosis of adipocytes, angiogenesis in AT, and lipid metabolism, with an emphasis on potential depot (subcutaneous vs visceral) differences and the impacts of obesity and T2D. This project will be performed totally in humans, their tissues and cells derived from those tissues. Insulin action in skeletal muscle will be studied both in vivo and in vitro in muscle tissue and cultured muscle cells and adipose tissue. Muscle and AT biopsies will be obtained for evaluation of the content of GRO, IL-8, and IL-15 mRNA and protein. Satellite cells from the biopsies will be propagated in culture and differentiated into myotubes to evaluate muscle specific production of the myokines of interest. Myotubes will also be treated with inflammatory and anti-inflammatory signals as well as individual myokines to determine if responsiveness to the factors varies with diabetes. Expression of the factors in myotubes will also be manipulated by genetic means. AT will be maintained in culture, treated with the proposed myokines of interest and both adipogenesis and apoptosis of mature adipocytes followed. AT will also be treated with individual myokines to follow effects on angiogenesis. Results from this project will provide molecular mechanisms for metabolic dysfunction seen in vivo in obesity and T2D and identify possible targets for therapeutic interventions. PUBLIC HEALTH RELEVANCE: Approximately 20% of the VA patient population is diabetic. A reasonable assumption is that an even greater portion is obese and display characteristics of the metabolic syndrome. Treatment of the glucose intolerance/insulin resistance and dyslipidemia prevalent in this population represents a crucial challenge to the VA Healthcare System. While metabolic dysfunction in diabetes involves multiple tissues, skeletal muscle is the major site of insulin resistance in thes individuals, and therefore a key target for therapeutic interventions. Muscle can also influence the behavior of other tissues. Understanding how changes in skeletal muscle function are transmitted to the whole body could lead to the design of interventions to augment the positive impacts of muscle-derived factors on glucose tolerance, fat metabolism and body composition. !